Ecology of Hymexazol-Insensitive Pythium Species in Field Soils

Mycopathologia 156: 333–342, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 333 Ecology of hymexazol-insensitive Pythium species in field soils Mohammed Ali-Shtayeh1 , Ayman M.A. Salah1 & Rana M. Jamous2 1 Department of Biology, An-Najah University; 2 Biodiversity and Environmental Research Center (BERC), Til Village, Nablus, Palestine Received 26 June 2001; accepted in final form 4 December 2002 Abstract Soils from 100 irrigated fields (95 under vegetables, 5 under citrus) in different geographical locations in the West Bank (Palestinian Autonomous Territory) were surveyed for hymexazol-insensitive (HIS) Pythium species using the surface soil dilution plate (SSDP) method with the VP3 medium amended with 50 mg/L hymexazol (HMI) (VP3H50), over a period of 12 months. HIS Pythium species were isolated from 37% of the soils surveyed, with mean population levels ranging from 4.3–1422 CFU g−1 dry weight. Eight HIS Pythium taxa were recovered on the VP3H50 medium, the most abundant of which was P. vexans (found in 29% of field soils surveyed). Seasonal variations in population levels of HIS Pythium species were studied in four fields over a period of 12 months. Significant seasonal variations in HIS population levels were detected in the four fields, with the highest population levels of HIS Pythium spp. encountered in spring and the lowest population levels in winter in three of the fields surveyed. Effects of HMI on linear growth and colony morphology of 149 Pythium ssp. isolates were examined on CMA amended with HMI at five concentrations. Pythium vexans isolates responded differently from those of the other Pythium species. Isolates of this important pathogen were more insensitive to HMI at high concentrations than the other main species tested. A large proportion of the P. ultimum isolates was either insensitive or weakly sensitive to HMI. Furthermore, a few isolates of other Pythium species were insensitive to the fungicide at various concentrations. The colony morphology of P. vexans isolates was not affected by HMI, whereas colonies of the other species showed sparse growth on the HMI amended medium relative to the control. The pathogenicity of P. vexans and P. ultimum isolates to cucumber seedlings was examined in growth chambers. Insensitive isolates of both species were found to be more virulent damping-off pathogens than the sensitive isolates. The present study demonstrates that HMI can not be used effectively in controlling Pythium spp. in soil inhabited with high densities of HIS Pythium spp. pathogens. Key words: hymexazol-insensitive Pythium species, pathogenicity, P. ultimum, P. vexans, seasonal variation, VP3H50 Introduction Hymexazol (5-hydroxy-5-methylisoxazol, HMI) (Sankyo Co., Ltd., Tokyo, Japan) and some of its derivatives are systemic fungicides. They have been used widely for the control of damping-off disease of many crops caused by Pythium species and some other soil-borne plant pathogens (e.g., Fusarium, and Rhizoctonia spp.) at relatively low concentrations. They were not effective in controlling Phytophthora species [1–3]. The selective toxicity of HMI toward Phytophthora and Pythium species, led Massago et al. [4] to suggest the inclusion of this fungicide in the selective media used for the isolation and quantification of Phytophthora species from soil to minimize the interference of Pythium on soil dilution plates. HMI inhibits the growth not only of Pythium spp., but also of Mortierella species [5]. It is inhibitory effect, however, increases when combined with other antimicrobial agents present in the selective media, such as rose Bengal and PCNB [5–7]. 334 Ho [8] reported different effects of HMI on growth and reproduction of some Phytophthora species, ranging between severe inhibition of some species to enhancement of growth of other species. As a result, Sneh & Katz [9] suggested the use of a reduced concentration of HMI (25 mg/L) in selective isolation media to enable recovery of HMI sensitive Phytophthora spp. from soil. Although HMI is used in selective media to prevent the overgrowth of Pythium spp., it is, however, clear that not all Pythium spp. are effectively inhibited by this fungicide [5, 9– 12]. Some species, such as P. vexans [11], and P. proliferum [12], are totally insensitive to HMI. They are very similar to Phytophthora spp. in this respect. Other species, such as, P. mamillatum, are insensitive at low concentrations (25 mg/L), but sensitive at higher concentrations [5]. Hymexazol-insensitive (HIS) isolates of Pythium species have been recently encountered frequently on selective media in soil dilution plates supplied with HMI at 50 mg/L [10, 13]. Furthermore, HIS isolates of P. ultimum have been found to be more virulent plant damping-off pathogens than the more hymexazolsensitive isolates (Ali-Shtayeh, unpublished data). HIS Pythium species appear to be a unique ecological group with respect to their response to fungicides, molecular features, and possibly virulence. Therefore, the present study was aimed at studying the ecology of this group of fungi in agricultural fields; and exploring any correlation between their insensitivity to HMI and other features including virulence and taxonomic status. Materials and methods Isolation and identification of HIS and other Pythium species from soil Sampling and sampling sites: One hundred irrigated fields (95 under vegetables, and 5 under citrus trees) distributed in the West Bank (Palestinian Autonomous Territory) were selected on the basis of cultural practices and geographical locations (Table 1). Four of these fields (2 in Nablus and 2 in Hebron areas) were sampled 6 times (once every other month) over a period of 12 months (June 97–May 98) to account for seasonal variation in Pythium spp. populations. The remaining fields were sampled only once. During each sampling time, four well-spaced soil samples, each approximately 250 g fresh weight, were taken from Table 1. Characteristics of the fields studied. Locality pH % Water % Organic matter Nablus (11 veg., citrus 1) Tulkarem (13 veg., 1 citrus) Jenin (13 veg.) Qalqilia (16 veg., 1 citrus) Jordan Valley (5 veg., 2 citrus) Ramallah (12 veg.) Bethlehem (13 veg.) Hebron (12 veg.) 7.1–7.8 7.3–7.7 7.1–8.3 7.1–7.8 7.3–8.1 7.3–7.7 7.1–7.9 7.2–7.4 15.4–37.5 15.4–26.0 24–37.1 12.2–35.6 9.5–33.0 18–34.8 13.2–30.1 17.6–28.9 0.8–6.4 0.5–3.4 0.4–4.2 1.6–7.4 1.4–4.2 3.8–9.8 1.6–4.9 0.5–3.9 an area of 16 m2 at a depth of 0–10 cm. The four samples were thoroughly mixed in a single plastic bag as a composite sample. Soil samples were processed on the day of collection. Soil characteristics including moisture content, pH, and organic matter were determined using standard methods [26, 35]. The remaining composite samples were divided into three equal parts each representing a replicate sample. Isolation of total and hymexazol-insensitive (HIS) Pythium species in field soil: The surface soil dilution plate method (SSDP) and the Vancomycin Pimaricin Pentachlorobenzene Penicillin (VP3) nutrient medium (agar 23 g, cornmeal agar 17 g, sucrose 20 g, CaCl2 10 mg, MgSO4 ·7H2 O, ZnCl2 1 mg, CuSO4 ·5H2 O 0.02 mg, MoO3 0.02 mg, MnCl2 0.02 mg, FeSO4 ·7H2 .O, 0.02 mg, thiamin HCl 100 µg, vancomycin 75 mg, pimaricin 5 mg, penicillin 50 mg, PCNB 100 mg, rose Bengal 2.5 mg) [16], and VP3 medium amended with HMI (Tachigaren, 99.5% purity) at 50 mg/L (VP3H50 medium) were used for the isolation of Pythium species from soil throughout this study. Soil dilutions of 1 : 50, 1 : 100 and/or 1 : 250 in sterile 0.08% water agar, depending on the anticipated Pythium population levels in the soil were used. One-ml of the suspension was pipetted on the surface of each plate, and spread over the surface using a sterile, bent glass rod. 1 : 50 soil dilution was usually used to isolate HIS Pythium species on the VP3H50 medium. Fifteen plates were used for each sample of soil (5/replicate soil sample). The plates were incubated in the dark at 22 ◦ C for 36–42 h. After 72 h incubation, final counts of the Pythium spp. colonies were recorded as the number of Pythium spp. colonies per plate. The mean number of the Pythium spp. colonies forming units per gram dry weight 335 Table 2. Pythium species isolates used in tests on the effect of hymexazol on the linear growth of Pythium species and in the pathogenicity experiments. Pythium species P. aphanidermatum P. oligandrum P. paroecandrum P. torulosum P. ultimum P. vexans Pythium sp.b Total Source Recovered in the present study FCCAUa Total 15 4 4 1 16 43 0 83 11 0 2 1 37 11 4 66 26 4 6 2 53 54 4 149 a FCCAU: Fungal Culture Collection of An-Najah University, An- Najah University, Nablus. b Unidentified, with lobulate sporangia and small plerotic oospores. (CFU g−1 D wt.) for each replicate soil sample was calculated as follows: CFU g−1 D. wt. = (total number of Pythium spp. colonies per replicate sample × dilution factor × soil sample fresh wt.)/(soil sample D. wt. × number of replicate plates). The %HIS Pythium isolates were calculated as follows: %HIS Pythium spp. isolates = (number of VP3H50 Pythium isolates/number of total Pythium colonies) × 100%. Identification of Pythium species: Identification of species was mainly based on monographs or keys of Plaats-Niterink [17], Ali-Shtayeh [18] and Dick [19]. Sensitivity to HMI Fungal isolates: One hundred and forty nine isolates of seven Pythium species P. aphanidermatum, P. oligandrum, P. paroecandrum, P. torulosum, P. ultimum, P. vexans, and P. sp. (unidentified with lobulate sporangia and small plerotic oospores) were tested for their susceptibility to HMI (Table 2). Of these isolates, 83 were isolated during this study and 66 were obtained from the Fungal Culture Collection of An-Najah University (FCCAU), Nablus. Effect of HMI on linear growth of Pythium species: Six concentrations (0, 10, 30, 50, 70, 90 mg/L) of HMI were used. HMI was dissolved in 2-ml distilled water and was added to the medium when it had cooled down to 50–55 ◦ C. Corn meal agar (CMA) medium was the base medium used in this study. Five-mm diam. agar discs were cut out from the periphery of a 48- h old colony of each isolate on CMA. One disc was placed in the center of each agar plate with the specified concentration of HMI. Two replicate plates were used for each isolate per treatment. The plates were incubated in the dark at 22 ◦ C for 48 hours. Colony radius was then measured in three different directions for each plate. The percentage of growth inhibition (% GI) was calculated for each isolate as follows: % GI = 100 - [(100% × colony radius on supplemented medium)/colony radius on CMA]. Pathogenicity of HIS Pythium isolates Test isolates: All isolates of P. ultimum and P. vexans (Table 2) were tested for their pathogenicity to cucumbers under growth chamber conditions. Preparation of mycelial inocula: The method of Martin [20] was used for inoculum preparation. A mixture of 3% (w/w) corn meal and sand with 20% water was used for the preparation of the initial inocula for each isolate. Five hundred grams of the corn mealsand mixture were placed in a 500-ml flask, covered with aluminum foil, and autoclaved at 121 ◦ C for 45 minutes. Initial inocula were prepared by aseptically adding five 5- mm diam. agar discs from actively growing colonies. The inocula were incubated under aseptic conditions at 24 ◦ C for 10 days. All flasks were shaken every 2 days to insure uniform colonization. The population density for each inoculum was determined by the SSDP method on the VP3 medium. The colonized mixture preparations were then used as inocula to inoculate the planting substrate to reach the desired density. Preparation of planting mixture: The planting mixture was prepared by mixing equal volumes of vermiculite and peatmoss. The mixture was autoclaved before inoculation with the Pythium spp. inocula. The concentrations of the inocula in the planting mixtures were 500, and 50 CFU g−1 for the P. vexans and P. ultimum isolates, respectively. Growth chamber experiments: All growth chamber experiments were conducted with plastic pots (1L volume). Three replicate pots were used per isolate. Soil was moistened and twenty cucumber seeds (Dalila) were planted in each pot at a depth of 1.5 cm. All pots were initially watered to saturation with distilled water. They then received watering as necessary. All pots were maintained at 20 ◦ C with 12 hours of light 336 per day cycle. The percentage of emergence was recorded after 10 days. The infection rate was calculated as follows: 100× (number of surviving seedlings of the control – number of surviving seedlings in the infested soil)/number of surviving seedlings of the control. All pathogenicity experiments were repeated twice. Statistical analysis: The correlation coefficients of soil factors and their presence or absence, and abundance (mean population levels) were used to detect any relationship between soil factors and the population levels of HIS Pythium species. Analysis of variance and Duncan’s multiple-range test were used to determine the significance of seasonal fluctuations. The data on sensitivity of the Pythium species to HMI were analyzed by using variance and cluster analyses. Multiple comparisons were used to compare the responses of the Pythium spp. isolates at the different concentrations of HMI. The ANOVA and Scheffee’s tests were used to detect differences among the various species in their susceptibility to HMI. The grouping of isolates in five categories of sensitivity was done by cluster analysis. This latter procedure was employed to reveal any similarities among the subjects that were measured, and to determine a classification scheme that accounted for variance among the subjects [21]. The relationship between virulence and sensitivity to HMI was determined by regression analysis. All statistical analysis tests were performed by using the SPSS computer program [22]. Results Occurrence and mean population levels of HIS Pythium species in soils: Vegetation type, pH, organic matter content, and moisture content of the hundred soil samples studied are given in Table 1. Summary data on the occurrence, and abundance (CFU g−1 D. wt.) of HIS- and total Pythium species recovered from the fields are given in Table 3. Pythium species were recovered from 88% of the fields sampled with mean population levels ranging from 0.0–1736 CFU g−1 D. wt. HIS Pythium species were detected in 37% of the soils using the VP3H50 medium. Mean population levels of HIS Pythium spp ranged from 0.0–1422 CFU g−1 D. wt. Eight taxa of Pythium were isolated on the VP3H50 medium. The most abundant HIS Pythium species was P. vexans; it was isolated from 29% of the fields. The next most fre- quently isolated HIS species was P. ultimum, detected in 4% of the fields. Mean population levels of HIS Pythium varied considerably between the different localities studied (Table 3), ranging from 0.6–176.5 CFU g−1 D. wt., with the Jenin locality yielding the highest mean population level (176.5), and the Jordan Valley yielding the lowest population level (0.6). The number of HIS Pythium taxa recovered from different localities ranged from 1–6 species (Table 3), with the Nablus locality yielding the highest number of HIS Pythium taxa (6), followed by Bethlehem and Hebron (4). Fluctuations in population levels of HIS Pythium species in field soils: Inoculum density levels of HIS Pythium species was determined in four field soils over a 12-month period using the VP3H50 medium. Inoculum density levels varied significantly (P = 0.001) with time throughout the course of the study in four fields (Table 4). HIS Pythium population levels followed almost similar seasonal fluctuation patterns with the highest population levels occurring in spring and early summer, and lowest in winter. Fluctuation in mean inoculum density levels of individual HIS Pythium species were also detected in all fields. In field (A) however, P. vexans was the only HIS Pythium species recovered. Other Pythium species including P. oligandrum, P. ultimum, and Pythium group G, were recovered from the remaining three fields (Table 4). Sensitivity of Pythium species to HMI: Data on the effect of HMI on linear growth were recorded as the percentage of mycelial inhibition of the Pythium species. Response curves of the percentages of growth inhibition against HMI concentrations for P. aphanidermatum, P. ultimum, and Pythium vexans are presented in Figure 1. Other species P. oligandrum, P. paroecandrum, P. torulosum, and P. sp., unidentified) were excluded from this figure since they were represented only by a few isolates (2–6). The mean percentage of mycelial growth inhibition (%GI) of all species tested is presented in Table 5. A linear relationship was detected between fungicide concentration and sensitivity of the Pythium species in terms of mycelial inhibition (correlation coefficient = 0.912). Single isolates of the Pythium species tested varied considerably in respect to their susceptibility to HMI. On the basis of their response to HMI at various concentrations, all isolates were grouped into five categories by cluster analysis (Table 6). Response curves 337 Table 3. Occurrence and range of means of population levels∗ of hymexazol-insensitive (HIS) Pythium species (CFU g−1 D. wt.) in soils in different localities in the West Bank. HIS Pythium spp. Population (CFU g−1 D. wt.) Total HIS Other HIS Pythium spp P. vexans Pythium spp Locality Bethlehem Hebron Jenin Jordan Valley Nablus Qalqilia Ramallah Tulkarm Frequency† 67.8 71.8 176.5 0.6 49.7 64.3 35.0 17.9 37 66.9 57.1(0.0–1422) 176.5 0.0 36.1 61.2 35.0 17.9 29 2.9∗∗∗∗∗ 14.7∗∗∗∗∗ 0.0 0.6∗∗∗∗ 8.9∗∗ 3.1∗∗∗ 0.0 0.0 11 %HIS Pythium spp Total Pythium population 594.7 548.7(0.0–1736) 265.6 60.3 371.2 275.4 426.5 295.8 88 0.0–38.9 0.0–36.5 0.0–100 0.0–20 0.0–36.6 0.0–61.0 0.0–15.8 0.0–32.2 ∗ Means of three replicate samples. Soils were sampled only once between June and July 1997. ∗∗ P. torulosum, P. paroecandrum, P. aphanidermatum, P. oligandrum, P. group G; ∗∗∗ P. ultimum; ∗∗∗∗ P. aph; ∗∗∗∗∗ P. ult, P. par, P. unidentified Pythium sp. † Percent of soils in which HIS and Pythium species were found. Table 4. Mean population levels (CFU g−1 D. wt.) of total and HIS Pythium species and seasonal distribution of HIS Pythium spp. in four fields in the West Bank. Fieldsa Date A HIS-vex (others)b Total Pythium B HIS-vex (others) Total Pythium C HIS-vex (others) Total Pythium D HIS-vex (others) Total Pythium June 97 Sep 97 Nov 97 Jan 98 Mar 98 May 98 Mean SD 56 (0) 50 (0) 96 (0) 126 (0) 156 (0) 117 (0) ± 100.6 ± 42.6∗ 306 318 593 623 810 401 508.3 ± 204.2∗ 277 (25) 105 (25) 68 (22) 94 (0) 142 (0) 154 (23) 140.0 ± 69.9∗∗ 892 511 277 287 792 754 585.3 ± 249.2∗∗ 507 (0) 119 (0) 120 (0) 129 (0) 151 (0) 349 (87) 229.0 ± 153.1∗∗ 1648 599 809 730 1450 2047 1213.8 ± 534.5∗∗ 105 (21) 65 (0) 43 (14) 82 (24) 136 (32) 943 (100) 228.9 ± 326.5∗∗ 353 235 218 395 553 2317 678.5 ± 741.1∗∗ a Sites A & B in Nablus area, and sites C & D in Hebron area. b Vex: Pythium vexans, others include P. ultimum, P. oligandrum, and P. group G. ∗ p < 0.01; ∗∗ p < 0.001. Table 5. Effect of hymexazol on mycelial growth of Pythium speciesa as a mean percentageb of mycelial growth inhibition (%GI) Fungal species P. aphanidermatum P. oligandrum P. paroecandrum P. torulosum P. ultimum P. vexans P. spc Hymexazol concentrations (µg/ml) 10 30 50 70 90 68 ± 11.5 73.3 ± 9.5 42.8 ± 22 14.5 ± 8.5 52.9 ± 21.3 52.9 ± 19.4 12.3 ± 5.1 92.6 ± 9.0 90.3 ± 4.0 76.0 ± 4.9 56.5 ± 2.5 85.5 ± 9.4 74.3 ± 24 69.3 ± 2.9 98.5 ± 0.0 98.8 ± 0.0 89.0 ± 5.3 84.5 ± 15.5 96.3 ± 7.1 83.7 ± 24.6 77.8 ± 3.2 79.9 ± 10.8 81.3 ± 5.8 57.8 ± 16.7 23 ± 0.5 66.7 ± 19.9 66.7 ± 21.8 36.8 ± 3.3 87.1 ± 11.8 87.0 ± 4.9 69.3 ± 10.4 45 ± 1 75.7 ± 11.9 64.6 ± 23.4 54.3 ± 5.9 a Number of isolates of each species as in Table 2. b Mean ± SD. c Unidentified, with lobulate sporangia and small plerotic oospores. 338 Figure 1. Response curves of the percentage of mycelial growth inhibition against hymexazole concentrations for three Pythium species. vex, P. vexans; ult, P. ultimum; aph, and P. aphanidermatum. (mean percentage inhibition vs HMI concentration) for the five categories are shown in Figure 2. Category A (resistant) included 8 isolates of P. vexans (Table 6). Isolates of this category were highly resistant to HMI at all concentrations tested. Category B (insensitive) included 15 isolates: of P. paroecandrum, 1 isolate; P. torulosum, 2 isolates; P. ultimum, 6 isolates (11.3%); P. vexans, 2 isolates (3.7%); and 4 isolates of the unidentified Pythium species. Isolates of this group were totally insensitive to HMI at concentrations of 10, 30 and 50 mg/L. They tolerated the higher concentrations (70 and 90 mg/L) with good growth. Category C (weakly sensitive) consisted of 32 isolates: 1 isolate (3.8) of P. aphanidermatum, 2 isolates of P. paroecandrum, 8 isolates (15.1) of P. ultimum, and 21 isolates (38.9) of P. vexans. Isolates of this group were classified as weakly sensitive to HMI; they were not sensitive at 10 and 30 mg/L, but were moderately sensitive at higher concentrations. Category D (moderately sensitive) included 66 isolates: P. aphanidermatum 12 isolate (46.2), P. paroecandrum 3 isolates, P. ultimum 32 isolates (60.4), and P. vexans 18 isolates (33.3). These isolates were moderately sensitive to HMI. They grow well on the medium amended with HMI 10–70 mg/L with a high percent of inhibition relative to the control. They were completely inhibited (>90% inhibition) at 90 mg/L HMI. Category E (sensitive) consisted of 28 isolates: P. aphanidermatum 13 isolates (50), P. oligandrum 2 isolates, P. ultimum 7 isolates (13.2), and P. vexans 5 isolates (9.3). These isolates were sensitive to HMI with growth inhibitions more than 50% at 10 and 30 mg/L HMI, and showed >85% inhibition at higher concentrations. Figure 2. Response curves of the percentage of mycelial growth inhibition against hymexazole concentrations for the five categories of Pythium spp. isolates. Categories: A, resistant; B, insensitive; C, weakly sensitive; D, moderately sensitive; E, sensitive. The species varied considerably (p = 0.001) in their response to HMI (Table 5, Figure 1). The overall picture of the three curves revealed that P. vexans was the least affected species (totally insensitive), followed by the isolates of P. ultimum, which were weakly sensitive at low concentrations, and moderately sensitive at higher concentrations, and P. aphanidermatum, which was relatively sensitive. Percent of growth inhibition ranged from 9–95, 46–100, and 67–100 at 50 mg/L HMI for isolates of P. vexans, P. ultimum, and P. aphanidermatum, respectively (Figure 1). The sensitivity (GI%) of the three species at various HMI concentrations (Figure 1) differed significantly according to Schefee’s test (p < 0.05). Pythium vexans was significantly less sensitive than the two species at all concentrations; it was totally insensitive to HMI. Pythium ultimum, was weakly to moderately sensitive, and differed considerably from P. aphanidermatum at 10, 30 and 50 mg/L (p = 0.05). The latter two species were, however, sensitive to HMI at higher concentrations (>50 mg/L). The response of the colony morphology of P. vexans isolates on HMI amended media was different from that of the other species. All P. vexans isolates from the resistant, insensitive and weakly sensitive groups grew well on HMI amended media with colony morphology similar to that of the control. However, in the HMI altered colony morphology of the majority of insensitive P. ultimum isolates; hyphal density was almost lower than that of the controls. Pathogenicity of the Pythium spp. isolates in relation to their sensitivity to HMI: Results of the pathogenicity (in terms of infection rates) experiments using 107 isolates of P. vexans and P. ultimum on cucumber seedlings revealed that the different isolates of the Pythium spp. produced significant decrease (p < 0.01) in seed- 339 Table 6. The distribution of the Pythium species isolates in the five categories with regard to their susceptibility to hymexazol. Categorya A (Resistant) B (Insensitive) C (Weakly sensitive) D (Moderately sensitive) E (Sensitive) Total Number of isolates from each species P. vexans P. ulimumt P. aphanidermatum 8 (14.8)b 2 (3.7) 21 (38.9) 18 (33.3) 5 (9.3) 54 (100) 0 (0) 6 (11.3) 8 (15.1) 32 (60.4) 7 (13.2) 53 (100) 0 (0) 0 (0) 1 (3.8) 12 (46.2) 13 (50) 26 (100) Other species Total 0 7 2 4 3 16 8 15 32 66 28 149 a Grouping of all isolates into the five categories was made by cluster analysis. b Values between parentheses represent the percentage of that category in the total number of isolates in the same column. ling emergence as compared to the control. Means of % damping-off given were 25% and 69% for isolates of P. vexans and P. ultimum, respectively. Significant, interspecific variations in the virulence of isolates were also detected (p < 0.05). The relationship between the virulence of the isolates of P. vexans and P. ultimum and their sensitivities to HMI is presented. A negative correlation was found between virulence and sensitivity (Table 7). Isolates with the lower sensitivity were generally more virulent than the more sensitive isolates. Discussion Our study clearly demonstrated that HIS Pythium spp. are widely distributed in field soils in the West Bank. Eight species of Pythium were recovered using the VP3H50 medium with P. vexans as the most frequently isolated species being found in 29% of the fields sampled. In fact, it was recovered from all soils, as demonstrated by its recovery on the VP3 plates. This indicated that resistance to HMI is probably a distinct character of this species as predicted by other workers [5, 11]. The other HIS Pythium species were, however, isolated at lower rates from some fields on the VP3H50 plates, including P. ultimum (4%), P. paroecandrum (3%), P. aphanidermatum (2%), and P. torulosum, P. oligandrum, and P. group G (1%). All these species were isolated at higher rates from soils on the VP3 medium plates in the current study. This is in agreement with previous studies carried out on the ecology of Pythium species in soils in the Palestinian area [16, 23, 24]. They demonstrated their widespread distribution in soil. The presence and abundance of HIS Pythium spp. was shown to be correlated with the higher moisture levels in the soil’s environment. This finding is similar to that found for Pythium species reported by several investigators [e.g., 16, 23–26]. However, no significant correlation was detected between the presence and abundance of HIS Pythium spp. and the soil’s organic matter content and pH (Table 8). Significant seasonal variations in the levels of HIS Pythium spp. population levels were detected in the fields studied. Generally, the highest CFU levels occurred in spring and early summer and lowest in winter. This may be attributed to variations in soil temperature, which is usually higher in spring and summer months than in winter, since all HIS Pythium species recovered from these fields had optimum temperatures of 25–27 ◦ C for growth [8]. On the other hand, the other Pythium species generally had their highest population levels in winter and early spring and lowest in summer. This is in agreement with that reported for total Pythium species from soils from the West Bank, Gaza and England studied over a period more than 12 months [27, 28]. The seasonal fluctuation of these species was partially associated with environmental factors [27]. However, soil moisture was not expected to account for seasonal variations in HIS Pythium population levels in the soil, since moisture content in the irrigated fields studied were relatively high with little variation, during the 12-month period of study. Radial growth on CMA often varied among the different species and among isolates of the same species. Their responses to the fungicide were not always similar at the various concentrations. Since inter- and intra-specific comparisons are an important part of the sensitivity experiment, the comparisons and constructions of response curves were based on percentage 340 Table 7. Correlation coefficients, obtained by regression analysis of infection rates of seedlings against the percent of growth inhibition at the various concentrations of hymexazol. Pynthim species Correlation coefficients at different hymexazol concentrations 10 30 50 70 90 P value P. vexans P. ultimum −0.352 −0.395 −0.416 −0.396 Table 8. Correlation coefficients obtained by regression analysis between soil factors and mean population levels of HIS Pythium spp. Variable Correlation coefficient P value % Soil moisture % Organic matter % Soil moisture + % organic matter pH 0.245 0.0722 0.244 0.0024 0.01 0.47 0.06 0.1 growth inhibitions relative to radial growth of the control. The growth of a few species of Pythium on a HMI-containing medium has been investigated [5, 11, 13]. However, in our study a large number of soil samples was tested for the presence of HIS Pythium species to be able to cover their variations in habitats as much as possible. Also large numbers of isolates of three Pythium species, which were suspected to be resistant, or hymexazol-tolerant, were tested for their response to the fungicide. The range of concentrations of the fungicide used was selected to give maximum information on the shape of the inhibition curves, and to enable intra- and inter-specific comparisons to be made. The sensitivity test responses of the Pythium spp. to the fungicide were notably diverse and could not be simply described as resistant or sensitive. The P. vexans isolates responded differently from the isolates of other species. The majority (31/54, 57%) of P. vexans isolates were resistant (14.8%), insensitive (3.7%) or weakly sensitive (38.9%). However, 18 isolates (33.3%) showed some level of tolerance to HMI at 10, 30 and 50 mg/L, and were classified as moderately sensitive. Only 5 isolates (9.3%) of P. vexans were classified as sensitive to the fungicide although the inhibition of these isolates was not complete. They were also recovered on VP3H50 plates. −0.442 −0.348 −0.367 −0.305 −0.367 −0.298 0.01 0.03 The isolates of P. ultimum showed diverse responses to the fungicide, but to a lesser degree than P. vexans. However, a large proportion (14/53, 26.4%) of this important pathogen’s isolates was either insensitive or weakly sensitive to the fungicide. Isolates of P. aphanidermatum were either moderately or totally sensitive to the fungicide at various concentrations, except one isolate that was classified as weakly sensitive. This isolate was one of two isolates of this species that were recovered on VP3H50 plates during our study. The variation in sensitivity to HMI of the different Pythium species may be have been due to differences in the efficiency of the defense mechanisms [11] of these species against the fungicide, including immobilization of the fungicide due to binding, inactivation or degradation of the active part of the fungicide molecule, and adaptive changes in the metabolism of the fungus exposed to the fungicide. Differences between the sensitive and resistant isolates of a fungus may also be due to the extent of degradation of the fungicide by the fungus [11]. The differential toxicity of the fungicide for certain species or isolates of soil-borne fungi may be due not only to its antimicrobial activity, but also to its metabolic alteration by the microorganisms and to the life form of the fungus affected [1]. Some isolates showed tolerance to HMI in the sensitivity experiment and were grouped with the insensitive isolates although they were not isolated on the VP3H50 plates. On the other hand, some VP3H50 isolates were inhibited at a high rate in the sensitivity tests. It is therefore important to assess the response of different life forms of the fungus to HMI. HMI has been used for the control of Pythium damping-off of many crops at relatively low concentrations [1–3]. However, the current study has shown that some Pythium species, some of which are well-known plant pathogens (e.g., P. vexans and P. ultimum), are resistant or insensitive to HMI. In ad- 341 dition, our study demonstrated that the HIS isolates were more virulent pathogens than the sensitive ones. The percentages of damping-off seedlings were larger in the planting mixtures infested with the HIS isolates. This may be partly attributed to differences in the abilities of the HMI-insensitive and sensitive isolates to produce celluloytic and pectolytic enzymes. It is important that fungicide mixtures and applications maximize crop protection and minimize damage to related species of fungi in the community, thereby causing the least disruption of the environment. Unfortunately, this principle may not be achieved when using HMI to control Pythium species. Excessive dependence on the use of HMI in fields inhabited with HIS Pythium will result in continued increases in the inoculum densities of these virulent pathogens. So HMI appears to be effective for disease control as long as the proportion of resistant isolates of the Pythium spp. in the soil is low, but its use should be discontinued in soils with high proportion of resistant Pythium spp. isolates. However, if HMI is used at high concentrations in controlling damping-off, it may cause retardation of the growth and germination of the seedlings [1]. The presence of HIS Pythium spp. at high rates in soils was always a problem in many studies aimed toward the isolation and quantification of pure cultures of Phytophthora spp. using HMI as the selective agent [10, 13]. Many Pythium spp. outgrew the growth of the Phytophthora species in HMI amended media [13]. In our study, HIS Pythium isolates had been recovered on VP3H50 plates from 37% of the soil samples tested. On the other hand, many Phytophthora spp. are sensitive to HMI [8, 9, 29]. So the use of HMI as a selective agent for the isolation and numeration of Phytophthora spp. in such soils may not be practical. Pythium vexans has some characteristics that distinguish it from the other Pythium species. These characteristics are based on morphological characters. It has bell-shaped antheridia [18]; and physiological characteristics, which include, the requirement of thiamin for growth [30]. Its response to some fungicides (e.g., metalaxyl and HMI) is different from the rest of the Pythium species [4, 11]. Furthermore, this species differs from other Pythium spp. in some molecular features. Its ribosomal DNA (rDNA) differs from that of the typical characters of the genus Pythium [31]. In ribosomal RNA (rRNA) sequencing, P. vexans again showed different sequences in five sites, and it was close to Phytophthora spp. in four of these sites [32]. 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Characterisation of a gene cluster of Phytophthora cryptogea, which codes for elicitins, proteins inducing a hypersensitive-like response in tobacco. Molec Plant-Microbe Interact 1995; 8: 996–1003. Address for correspondence: M.S. Ali-Shtayeh, Department of Biologic Science, An Najah University, PO Box 696, Nablus Israel Phone: 0097292346406; E-mail: [email protected]